A new indocyanine green fluorescence lymphography protocol for identification of the lymphatic drainage pathway for patients with breast cancer-related lymphoedema

Breast cancer related lymphoedema (BCRL) is a common side effect of cancer treatment. Recently indocyanine green (ICG) fluorescent lymphography has become a popular method for imaging the lymphatics, however there are no standard protocols nor imaging criteria.

T E C H N I C A L A D V A N C E Open Access

A new indocyanine green fluorescence

lymphography protocol for identification of

the lymphatic drainage pathway for patients

with breast cancer-related lymphoedema

Hiroo Suami1* , Asha Heydon-White1, Helen Mackie1,2, Sharon Czerniec1, Louise Koelmeyer1and John Boyages1

Abstract

Background: Breast cancer related lymphoedema (BCRL) is a common side effect of cancer treatment Recently indocyanine green (ICG) fluorescent lymphography has become a popular method for imaging the lymphatics, however there are no standard protocols nor imaging criteria We have developed a prospective protocol to aid in the diagnosis and therapeutic management of BCRL

Methods: Lymphatic imaging procedures were conducted in three phases Following initial observation of

spontaneous movement of ICG in phase one, manual lymphatic drainage (MLD) massage was applied to facilitate ICG transit via the lymphatics in phase two All imaging data was collected in phase three Continuous lymphatic imaging of the upper limb was conducted for approximately an hour and lymphatic drainage pathways were determined Correlations between the drainage pathway and MD Anderson Cancer Centre (MDACC) ICG

lymphoedema stage were investigated

Results: One hundred and three upper limbs with BCRL were assessed with this new protocol Despite most of the patients having undergone axillary node dissection, the ipsilateral axilla drainage pathway was the most common (67% of upper limbs) We found drainage to the ipsilateral axilla decreased as MDACC stage increased Our results suggest that the axillary pathway remained patent for over two-thirds of patients, rather than completely

obstructed as conventionally thought to be the case for BCRL

Conclusions: We developed a new ICG lymphography protocol for diagnosing BCRL focusing on identification of

will allow a personalised approach to manual lymphatic drainage massage and potentially surgery

Keywords: Lymphoedema, Lymphography, Lymphatic system, Manual lymphatic drainage, Breast cancer,

Molecular imaging

Background

Breast-cancer related lymphoedema (BCRL) is a common

side effect of cancer treatment causing physical, functional,

psychological and financial challenges for individuals and

impacting their quality of life [1–4] Lymphoscintigraphy is

the standard technique in lymphatic imaging for diagnosing

lymphoedema [5,6] Although no universal protocol exists, three imaging criteria are used in diagnosis; delayed transit

of radioactive tracer compared to the unaffected limb and presence of dermal backflow, the accumulation of the tracer

in the dermal lymphatics and absence or reduced number

of draining lymph nodes

Recently, Indocyanine Green (ICG) lymphography has become an alternate popular method for imaging the lymphatics ICG lymphography was initially used for breast sentinel node biopsy [7] Its application then extended to

© The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

* Correspondence: hiroo.suami@mq.edu.au

1 Australian Lymphoedema Education Research Treatment (ALERT) Program,

Faculty of Medicine and Health Sciences, Macquarie University, Level 1, 75

Talavera Rd, Sydney, NSW 2109, Australia

Full list of author information is available at the end of the article

lymphoedema diagnosis and mapping lymphatic vessels

prior to lymphovenous anastomosis (LVA) surgery [8–10]

The above lymphoscintigraphy criteria for lymphoedema

diagnosis cannot be applicable for ICG lymphography

be-cause penetration of the near infrared rays is limited to 2

cm from the skin surface making it difficult and

inconsist-ent to idinconsist-entify lymph nodes [11] However, ICG

lymphog-raphy has some advantages for lymphatic imaging over

lymphoscintigraphy ICG is a water-based solution and

therefore travels faster via the lymphatics compared to

technetium 99 m-sulfer colloid commonly used as the

radionuclide for lymphoscintigraphy, enabling high

reso-lution, and real time imaging Furthermore, ICG is not

radioactive and does not require special storage

precau-tions [12]

Due to the requirement of new imaging criteria for the

diagnosis of lymphoedema using ICG lymphography, we

have developed a prospective protocol to aid in the

diag-nosis of BCRL, assist decision making for therapeutic

management including ICG-directed manual lymphatic

drainage (MLD) massage and define selection criteria for

surgical options The aim of this study to summarise

ini-tial findings obtained by the new ICG lymphography

protocol in breast cancer related lymphoedema

Methods

A retrospective cohort audit was conducted, reviewing

prospectively collected data from patients with BCRL

who underwent ICG lymphography at the Australian

Lymphoedema Education, Research and Treatment

(ALERT) clinic at Macquarie University (MQ) between

February 2017 and April 2018 Data were sourced from

elec-tronic medical records and this audit was approved by MQ

Health Ethics Committee (Reference: MQCIA2018017)

Written informed consent was obtained from all patients in

this study

In three patients for the pilot study, we repeated the

ICG imaging after 24 h and compared with the images

obtained with this protocol If the patients had previous

lymphoscintigraphy in the affected limb, both

lymphoscin-tigraphy and ICG lymphography images were compared

ALERT ICG lymphography imaging protocol

The near infrared camera system (PDE Neo II; Hamamatsu

Photonics K.K.) was used for this study Indocyanine Green

(Verdye 25 mg; Diagnostic Green GmbH) was mixed with

5 ml of saline Four injection sites were used in the distal

aspect of the upper limb on the affected side: first and

fourth web spaces and ulnar and radial volar wrist regions

(Fig.1) These circumferential injection sites were chosen

based on our previous cadaveric lymphatic anatomy

stud-ies which demonstrated that lymphatic vessels originate

individually and have few interconnections [13–15] ICG

lymphography was only applied for the affected limb

instead of imaging the unaffected limb as a control because our cadaveric studies confirmed uniformity of the lymph-atic drainage pathways in normal anatomy between indi-viduals [13–15] Further we considered bilateral imaging to

be more costly, time-consuming and more stressful for the patient

Intradermal injections were performed with a 30-gauge needle and a 1 ml syringe At each injection site 0.05-0.1

ml (0.25-0.5 mg) of ICG solution was administered A cryogenic numbing device (CoolSense; CoolSense Medical Ltd.) was used immediately before each injection to re-duce needle discomfort [16]

Lymphatic scanning using the near infrared camera com-menced immediately after the injections and imaging data was recorded using a digital video recorder (MDR-600HD: Ikegami Tsushinki Co., Ltd.) Lymphatic imaging of the upper limb was continuously conducted in each upper limb for approximately an hour

Imaging procedures

Lymphatic scanning was conducted in three phases

Phase one

Observation of any spontaneous movement of ICG via the lymphatics for approximately 10 min Patients were encouraged to clench and unclench their hand ten times

to facilitate lymphatic uptake of the ICG

Phase two

Manual lymphatic drainage (MLD) massage was then performed by an accredited lymphoedema therapist to facilitate ICG transit via the lymphatics This MLD is undertaken by the therapist and the patient’s real time visualisation of the lymphatic vessels and areas of dermal backflow provides patient feedback of direction, speed and skin pressure required to move the ICG dye Scan-ning focused on identifying lymphatic vessels and the competency of their valves, direction of dermal backflow extension, and identifying lymph nodes We found that MLD facilitated dye movement more efficiently com-pared to post-injection exercise and delayed scanning although this was not formally evaluated When lymph-atic vessels were identified, their course was marked on the patient’s skin with a coloured pen (Fig 1a) Phase two continued until the dissemination of ICG reached a plateau without any further movement, usually between

30 and 45 min

Phase three

Demarcation lines of dermal backflow were marked on the skin, and collection of imaging data through still photography with both near infrared and digital cameras were taken (Fig 2 left and centre, and 3) Phase three takes approximately 15 min

Imaging data analysis

Still ICG images were montaged with image editing

soft-ware (Photoshop CC; Adobe Systems) to provide an

image of the whole upper limb The lymphatics were

designated into two categories; lymphatic vessels in the

subcutaneous tissue and dermal backflow which is reflux

of lymph fluid into dermal lymphatics Lymphoedema

was diagnosed by the presence of dermal backflow

Although ICG lymphography was considered mainly for

imaging the superficial lymphatics, the following

obser-vation helped us to interpret the images For example, if

the epitrochlear lymph node was identified in the medial

elbow, the efferent lymphatic vessel of the node was

known to run along the brachial artery in the upper arm

[17] Although the ICG near infrared signal in the upper

arm was often missing, we could identify the signal in

the operated axilla because of reduced soft tissue Thus

we could confirm that the lymphatic drainage pathway

drained to the ipsilateral axilla

Lymphatic drainage pathways were determined by the location of identified lymph nodes or extension of ICG

to the skin regions via dermal backflow or lymphatic vessels where lymph nodes were located underneath Lymphoedematous upper limbs were also classified by MDACC stage as 0: normal lymphatics, Stage 1: many patent lymphatic vessels with minimal patchy dermal backflow, Stage 2: moderate number of patent lymphatic vessels with segmental dermal backflow, Stage 3: few patent lymphatic vessels with extensive dermal backflow involving the entire arm, Stage 4: no patent lymphatic vessels seen with dermal backflow involving the entire arm with extension to the dorsum of the hand and Stage 5: ICG does not move from injection sites [18,19]

Results

One hundred and seven upper limbs at-risk or affected by BCRL were examined in 103 patients (unilateral: 99, bilat-eral: 4) Three patients who were previously diagnosed with Fig 1 ICG injection sites

Fig 2 Comparison of ICG lymphography and tracing photo (left and centre) and Lymphoscintigraphy image (right) in the same patient

unilateral BCRL were found by our imaging criteria to

demonstrate normal lymphatics (MDACC Stage 0) and an

additional bilateral patient who had normal lymphatics on

the side of the sentinel node biopsy were excluded Our

study cohort therefore consisted of 103 upper limbs

(uni-lateral 97, bi(uni-lateral 3) examined in 100 patients Patient

characteristics are described in Table 1 Of note, axillary

dissection was performed in 99 limbs, 2 had a sentinel

node biopsy and for 2 patients the extent of axillary surgery

was unknown

ICG lymphography findings

We could frequently specify sites in the upper limb where

the lymphatic vessel was obstructed Dermal backflow was

identified at these obstruction sites extending through the

dermal lymphatics (Additional file1: Video S1)

ICG lymphography demonstrated that MLD could

fa-cilitate transit of ICG via dermal backflow and lymphatic

vessels (Additional file2: Video S2 and Additional file3:

Video S3) ICG moved slower in dermal backflow and

faster in the lymphatic vessel probably due to the calibre

and contractility of the lymphatic vessels When MLD

was performed to areas of dermal backflow, ICG moved

directionally instead of extending in all directions We

recognised two patterns of the directional ICG spread in

dermal backflow First, dermal backflow was observed

initially at a site of lymphatic vessel obstruction and

moved towards an adjacent patent lymphatic vessel

Dermal backflow worked as a detour route acting as a

bridge between the obstructed and patent lymphatic

vessels Second, when no patent vessels remained in the

limb, dermal backflow extended directly to a lymph node

region such as the ipsilateral axilla, clavicular or

para-sternal regions

We also defined the demarcation line of dermal back-flow at the end of the procedure (Fig.1a in red) Of the three patients who underwent both ICG lymphography and lymphoscintigraphy there was good consistency of the presence of dermal backflow, identification of lymph nodes and lymphatic drainage pathways between the two techniques However, real-time ICG lymphography allowed precise demarcation of dermal backflow

In three patients who repeated the ICG imaging after

24 h, we found that, although the delayed-images had faded slightly, the demarcation lines of the dermal back-flow and the lymphatic drainage pathways were identical This suggests that our protocol of an approximately one hour ICG imaging session combined with MLD is suffi-cient to gain maximum information for patients with BCRL

Of the103 upper limb examined patients, none were classified into MDACC Stage 5 34 upper limbs (32%) demonstrated more than one drainage pathway Varia-tions of drainage pathway patterns are summarised in Table2and Fig.3 Overall the percentage of drainage to the ipsilateral axilla was 67% We found drainage to the ipsilateral axilla decreased as MDACC stage increased

As drainage to the axilla decreased by stage, we found drainage to the ipsilateral clavicular pathway increased reaching a peak of 55% for patients with MDACC Stage

3 lymphoedema Patients with MDACC Stage 4 lym-phoedema had the highest rate of drainage to the para-sternal pathway and contralateral axilla (17%) In these cases, dermal backflow in the upper limb extended to the anterior midline of the chest and rerouted to the contralateral axilla via the intact lymphatic vessels in the contralateral breast Of note, if there was a functional pathway to the proximal region of the ipsilateral upper limb, ICG did not extend beyond this region For example, dermal backflow extended either to the para-sternal region or to the contralateral axilla only when the pathway to the ipsilateral axilla or clavicular region was obstructed

Discussion

The diagnosis of lymphoedema is often difficult by phys-ical examination alone, especially in early stages Patients with BCRL often complain, for example, of discomfort

in specific areas of their upper limb instead of uniform changes or swelling in the whole limb In this study, we introduced a new ICG lymphography protocol for the upper limb to help to identify areas with underlying anatomical changes that occur in lymphoedema Our previous review study found that ICG lymphography had the potential benefit to elucidate the relationship between lymphatic drainage pathway and severity of lymphoedema [20] The drainage pathway to the clavicu-lar region was commonly identified for patients with

Table 1 Patient characteristics

Characteristic

Time since cancer diagnosis (yr) mean (SD) 7.26 (±6.88)

Breast Surgery type n (%)

Axilla surgery type n (%)

Adjuvant therapy n (%)

WLE Wide Local Excision, NSM Nipple Sparing Mastectomy, ALND Axillary

Lymph Node Dissection, SNB Sentinel Node Biopsy, yr year, n number

MDACC ICG Stage 2 or 3 lymphoedema and occurred

in 52-55% of patients respectively It was apparent that

sternal and contralateral pathway groups were found in

Stage 4 lymphoedema (Table2)

Lymphoscintigraphy has been the standard imaging

examination for lymphoedema [21, 22] However,

con-ventional lymphoscintigraphy protocols for

lymphoe-dema do not include identification of the lymphatic

drainage pathways because spontaneous transit of vis-cous radionuclide tracer cannot reach lymph nodes in lymphoedematous limbs constantly In recent, stress-lymphoscintigraphy including exercise was introduced

to improve lymphatic visualization but radiation ex-posure prevents applying MLD for facilitating tracer transit [23,24] In comparison ICG mixture moves fas-ter than the lymphoscintigraphy tracer and facilitation

Table 2 Lymphatic Drainage Pathways

ICG drainage pathways

MDACC stage No Ipsilateral axilla Clavicular Parasternal Contralateral axilla Ipsilateral Inguinal Unknown

Fig 3 Patterns of drainage pathway in ICG lymphography images (left) and tracing photos (right): a ipsilateral axilla, b clavicular, c parasternal, and d contralateral axilla

of ICG transit with MLD can reduce examination time

to specify the lymphatic drainage pathway and

pro-vides additional direct therapeutic guidance to the

patient and the therapist

Another advantage of ICG lymphography is that some

patients may indeed not suffer from lymphoedema In our

study four limbs in four patients were diagnosed as not

having BCRL as they had normal lymphatic drainage

with-out any dermal backflow Future research should address

the correlation of ICG lymphography with subclinical

lymphedema detected by bioimpedance spectroscopy

Further, there is often a misconception that lymphatic

drainage occurs away from the dissected axilla In Abe’s

lymphangiography studies 13 of 19 patients (68%) showed

patent lymph vessels passing through the axilla [25] This

was almost identical to our rate of 67% indicating that the

ipsilateral axilla is still considered as a vital pathway

Conventionally, BCRL was thought to be caused by

the complete obstruction of the lymphatic drainage to

the ipsilateral axilla secondary to surgical and/or

radi-ation intervention Our results contradict this notion

and suggest that the axillary pathway was restricted

functionally instead of complete obstruction in over

two-thirds of patients

Conclusion

We developed a new ICG lymphography protocol for

diagnosing BCRL focusing on identification of an

individ-ual patient’s lymphatic drainage pathway after lymph node

surgery to guide MLD and to assist with selection criteria

for lymphatic microsurgery ICG imaging combined with

MLD will allow a personalised approach to lymphoedema

care

Supplementary information

Supplementary information accompanies this paper at https://doi.org/10.

1186/s12885-019-6192-1

Additional file 1 Dermal backflow was identified with ICG lymphography.

Additional file 2 Gentle MLD could move ICG via lymphatic vessels in

mild lymphoedema.

Additional file 3 Firmer MLD could move ICG via dermal backflow in

severe lymphoedema.

Abbreviations

ALERT: Australian lymphoedema education, research, and treatment;

BCRL: Breast cancer related lymphoedema; ICG: Indocyanine green;

MDACC: MD Anderson Cancer Centre; MLD: Manual lymphatic drainage

Acknowledgements

The authors thank Philippa Sutton for editorial assistance with the

preparation of this article.

Authors ’ contributions

HS and AHW conceived and designed the protocol JB supervised the design

of the data form HS, AHW, and HM collected the data SC contributed to

the data analyses LK and JB supervised the project HS took the lead in

writing the manuscript All authors provided critical feedback and helped the

research, analysis and manuscript All authors read and approved the final manuscript.

Funding The authors state that this work has not received any funding.

Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate Data were sourced from electronic medical records and this audit was approved by MQ Health Ethics Committee (Reference: MQCIA2018017) Written informed consent was obtained from all patients in this study Consent for publication

Signed institutional consent form was obtained from each patient Competing interests

The authors declare that they have no competing interests.

Author details

1 Australian Lymphoedema Education Research Treatment (ALERT) Program, Faculty of Medicine and Health Sciences, Macquarie University, Level 1, 75 Talavera Rd, Sydney, NSW 2109, Australia.2Mt Wilga Private Hospital, Hornsby, NSW, Australia.

Received: 3 March 2019 Accepted: 23 September 2019

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